Method for obtaining information of test substance

ABSTRACT

Disclosed is a method for obtaining information of a test substance, the method including: forming a complex by causing a capture substance to bind to a test substance in a specimen; selectively collecting at least the complex from the specimen; immobilizing the complex collected from the specimen, onto a base plate; and obtaining information regarding a structure of the test substance from the complex immobilized on the base plate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Japanese Patent ApplicationNo. 2017-079793, filed on Apr. 13, 2017, entitled “METHOD FOR OBTAININGINFORMATION OF TEST SUBSTANCE”, the entire content of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an information obtaining method forobtaining information from a test substance.

BACKGROUND

Obtaining information regarding the structure of a test substance in asample is useful in pathological diagnosis and determination of anadministration policy. For example, in the case of Alzheimer's disease,the structure (size, length, aspect ratio, etc.) of the test substancesuch as amyloid β changes in accordance with progression of the disease.Thus, if information regarding the structure is obtained from the testsubstance, the disease condition can be properly understood. Examples ofdiseases caused by denaturation of protein include Huntington's disease,Parkinson's disease, prion, and ALS (amyotrophic lateral sclerosis), inaddition to Alzheimer's disease.

“Super-resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Toolfor Alzheimer's Disease Diagnostics” by William I. Zhang and five otherauthors, Journal of Alzheimer's Disease 46, Mar. 27, 2015, pp. 1007-1020(hereinafter, referred to as “Non-Patent Literature 1”) describes thefollowing: as shown in FIG. 19A, CSF (cerebrospinal fluid) is suppliedon a glass base plate 200 to cause an amyloid β 211 as a test substanceto be physically adsorbed; then, as shown in FIG. 19B, the labeledantibody 230 is caused to bind to the amyloid β 211 through a primaryantibody 220; and an image is detected by a super-resolution microscope.

According to the technique of Non-Patent Literature 1, as shown in FIG.19A, not only the amyloid β 211 but also an impurity 212 attach to theglass base plate 200. In some cases, the primary antibody 220 and thelabeled antibody 230 nonspecifically bind also to the impurity 212attached to the glass base plate 200. In such a case, as shown in FIG.19B, the impurity 212 is labeled with the labeled antibody 230. Further,in some cases, the labeled antibody 230 nonspecifically binds to theglass base plate 200. In such a case, as shown in FIG. 19B, a portion ofthe glass base plate 200 is labeled with the labeled antibody 230. Thus,when not only the amyloid β 211 but also the impurity 212, the glassbase plate 200, and the like are labeled with the labeled antibody 230,information regarding the structure of the amyloid β 211 as a testsubstance cannot be accurately obtained.

SUMMARY OF THE INVENTION

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary.

A first mode of the present invention relates to a method for obtaininginformation of a test substance. A method for obtaining information of atest substance according to the present mode includes: forming (S11 toS13, S21, S22) a complex (60, 61) by causing a capture substance (30,40, 50) to bind to a test substance (11) in a specimen (10); selectivelycollecting (S14, S15, S23, S24) at least the complex (60, 61) from thespecimen (10); immobilizing (S16, S26) the complex (60, 61) collectedfrom the specimen (10), onto a base plate (80); and obtaining (S17, S27)information regarding a structure of the test substance (11) from thecomplex (60) immobilized on the base plate (80).

The specimen is a liquid taken from a living body, and is CSF(cerebrospinal fluid), blood, plasma, interstitial fluid, or urine, forexample. The test substance is a cell, a polypeptide, a protein, anucleic acid, an exosome, a carbohydrate chain, or the like, and in themethod for obtaining the information of the test substance according tothe present mode, a multimer is appropriate. The test substance is anamyloid β oligomer which is made up of polymerized amyloid β monomers, atau oligomer which is made up of polymerized tau proteins, or the like.“Selectively collecting the complex” is not limited to collecting thecomplex only, but is a concept that encompasses collecting the complexwith substances other than the complex also slightly included. The baseplate is formed as a glass plate or the like, for example. The“information regarding the structure of the test substance” can broadlyencompass the size (e.g., length), the morphology (e.g. aspect ratio),the structure (e.g., the primary structure, the secondary structure, thetertiary structure, and the quaternary structure of protein), thechemical bond, the aggregation degree, and the like.

In the method for obtaining the information of the test substanceaccording to the present mode, the complex is selectively collected fromthe specimen and the collected complex is immobilized on the base plate.At this time, impurities such as substances other than the testsubstance contained in the specimen and the capture substance that hasnot formed the complex are separated from the complex and are suppressedfrom being transferred to the base plate. Therefore, to the base plate,substantially only the test substance is transferred and immobilized.Thus, the information regarding the structure of the test substance canbe accurately obtained.

In the method for obtaining the information of the test substanceaccording to the present mode, in the obtaining (S17, S27) of theinformation regarding the structure of the test substance (11), at leastone of size, morphology, structure, and aggregation degree of the testsubstance (11) is obtained.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance includes a capturesubstance (40) for labeling the test substance (11) with fluorescence.Even when this capture substance has not formed the complex, thiscapture substance is suppressed from being transferred to the baseplate, and thus, this capture substance can be suppressed from causingnoise.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance includes a capturesubstance (30) capable of binding to a solid phase (20). Even when thiscapture substance has not formed the complex, this capture substance issuppressed from being transferred to the base plate, and thus, thiscapture substance can be suppressed from causing noise.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance includes a capturesubstance (50) capable of binding to the base plate (80). Even when thiscapture substance has not formed the complex, this capture substance issuppressed from being transferred to the base plate, and thus, thiscapture substance can be suppressed from causing noise.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance (40) for labelingthe test substance (11) with fluorescence includes an antibody (42)labeled with a fluorescent dye (41), and in the forming (S13, S22) ofthe complex (60, 61), the antibody (42) labeled with the fluorescent dye(41) is caused to bind to the test substance (11). Accordingly, the baseplate can be suppressed from being directly fluorescence-labeled, andthus, the information regarding the structure of the test substance canbe accurately obtained.

In the method for obtaining the information of the test substanceaccording to the present mode, after the solid phase (20) is caused tobind to the complex (60, 61) through the capture substance (30) capableof binding to the solid phase (20), the solid phase (20) is selectivelyseparated (S14, S23) in the collecting (S14, S15, S23, S24) of thecomplex (60, 61) from the specimen (10). Accordingly, selectivelycollecting the complex from the specimen is facilitated. In addition, inthe latter stage, the complex can be easily detached from the solidphase.

In this case, in the collecting (S14, S15, S23, S24) of the complex (60,61) from the specimen (10), the complex (60, 61) is caused to bedetached (S15, S24) from the solid phase (20) after the solid phase (20)is selectively separated. Accordingly, since the solid phase is nottransferred to the base plate, the information regarding the structureof the test substance can be more accurately obtained.

The solid phase (20) includes a magnetic particle (21), and the solidphase (20) is selectively separated by attracting the magnetic particle(21) by magnetic force. Accordingly, the complex and the impurity can besmoothly separated from each other.

The capture substance (30) capable of binding to the solid phase (20)includes an antibody (32) which binds to the test substance (11) and asecond binding substance (31) which binds to the solid phase (20), andthe solid phase (20) includes a second binding partner (22) whichspecifically binds to the second binding substance (31). Binding betweenthe complex (60, 61) and the solid phase (20) is realized by bindingbetween the test substance (11) and the antibody (32) of the capturesubstance (30) capable of binding to the solid phase (20), and byspecific binding between the second binding substance (31) and thesecond binding partner (22).

In this case, a combination of the second binding substance (31) and thesecond binding partner (22) is selected from combinations of: an antigenand an antibody thereto; a ligand and a receptor therefor; anoligonucleotide and a complementary strand thereof; and biotinsincluding biotin and biotin analogs such as desthiobiotin and avidinsincluding avidin and avidin analogs such as streptavidin. Accordingly,the second binding substance and the second binding partner can bestably bound to each other.

The second binding substance (31) is an anti-hapten, and the secondbinding partner (22) is an anti-hapten antibody. Also in this case, thesecond binding substance and the second binding partner can be stablybound to each other.

In this case, the second binding substance (31) is a dinitrophenyl groupand the second binding partner (22) is an anti-dinitrophenyl groupantibody. Accordingly, the second binding substance and the secondbinding partner can be easily detached from each other.

In this case, the second binding substance (31) and the second bindingpartner (22) bound to each other are caused to be detached from eachother by use of a dinitrophenyl amino acid.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance includes a capturesubstance (50) capable of binding to the base plate (80) and a capturesubstance (30) capable of binding to a solid phase (20), and the capturesubstance (50) capable of binding to the base plate (80) and the capturesubstance (30) capable of binding to the solid phase (20) are differentfrom each other. In this case, two kinds of capture substancesindividually bind to the test substance. If the capture substance forimmobilizing the test substance onto the base plate and the capturesubstance for binding the test substance to the solid phase aredifferent from each other, the latter capture substance that has notbound to the test substance can be removed during collection of thecomplex. Thus, the capture substance capable of binding to the baseplate and not having bound to the test substance can be suppressed frombeing immobilized onto the base plate, and thus, immobilization of thetest substance onto the base plate can be more smoothly performed.

In this case, the capture substance (50) capable of binding to the baseplate (80) and the capture substance (30) capable of binding to thesolid phase (20) are substantially simultaneously put into the specimen(10). Accordingly, the two capture substances can be smoothly caused tobe bound to the binding site of the test substance.

In the method for obtaining the information of the test substanceaccording to the present mode, in the collecting (S14, S15, S23, S24) ofthe complex (60, 61) from the specimen (10), the complex (60, 61) isseparated from an impurity (13) on the basis of at least one of adifference in specific gravity, a difference in size, a difference inelectrical property, and a difference in immunoreaction between thecomplex (60, 61) and the impurity (13). The difference in size, thedifference in electrical property, and the difference in immunoreactionare, for example, those in gel filtration, electrophoresis, andimmunoreaction, respectively. Accordingly, appropriate separation can beperformed.

In the method for obtaining the information of the test substanceaccording to the present mode, the capture substance (50) capable ofbinding to the base plate (80) includes an antibody (52) which binds tothe test substance (11) and a binding substance (51) which binds to thebase plate (80), and the base plate (80) includes a binding partner (81)which specifically binds to the binding substance (51). After theantibody (52) included in the capture substance (50) capable of bindingto the base plate (80) is caused to bind to the test substance (11), thecomplex (60) is immobilized on the base plate (80) through specificbinding between the binding partner (81) and the binding substance (51),in the immobilizing (S16, S26) of the complex (60) onto the base plate(80). Accordingly, the test substance can be specifically bound to thebase plate not through physical adsorption but through mediation by thecapture substance. Thus, the test substance can be stably immobilized onthe base plate while the impurity is suppressed from being transferredto the base plate.

In this case, a combination of the binding substance (51) and thebinding partner (81) is selected from combinations of: an antigen and anantibody thereto; a ligand and a receptor therefor; an oligonucleotideand a complementary strand thereof; and biotins including biotin andbiotin analogs such as desthiobiotin and avidins including avidin andavidin analogs such as streptavidin. Accordingly, the binding substanceand the binding partner can be stably bound to each other.

The binding substance (51) is a type of the biotins, and the bindingpartner (81) is a type of the avidins. Accordingly, due to highaffinity, the test substance can be more stably immobilized on the baseplate.

In the method for obtaining the information of the test substanceaccording to the present mode, after the collecting of the complex (61)from the specimen (10), a capture substance (50) capable of binding tothe base plate (80) is caused to bind to the test substance (11).

In the method for obtaining the information of the test substanceaccording to the present mode, in the obtaining (S17, S27) of theinformation regarding the structure of the test substance (11), an imageof the test substance (11) is obtained by performing image capturing ofthe test substance (11) on the base plate (80). Accordingly, theinformation regarding the structure of the test substance can beobtained on the basis of the obtained image.

In the method for obtaining the information of the test substanceaccording to the present mode, in the collecting of the complex (60, 61)from the specimen (10), the test substance (11) and an impurity (13) areseparated from each other, and the test substance (11) is a protein as atest target contained in the specimen (10) and the impurity (13)includes a protein other than the protein as the test target.

In the method for obtaining the information of the test substanceaccording to the present mode, the test substance (11) is amyloid β. Inthis case, if the information regarding the structure of amyloid β isobtained, the obtained information can be helpful in diagnosis ofAlzheimer's disease and determination of an administration policy, forexample.

In the method for obtaining the information of the test substanceaccording to the present mode, the specimen (10) is cerebrospinal fluid.

In the method for obtaining the information of the test substanceaccording to the present mode, the obtaining (S17, S27) of theinformation regarding the structure of the test substance (11) isperformed by means of a microscope.

In this case, the microscope is a fluorescence microscope, a Ramanmicroscope, a probe microscope, or an electron microscope.

In the method for obtaining the information of the test substanceaccording to the present mode, the obtaining (S17, S27) of theinformation regarding the structure of the test substance (11) isperformed by means of a microscope having a resolution exceeding adiffraction limit of light. Accordingly, the information regarding thestructure of the test substance can be obtained at a resolutionexceeding the diffraction limit of light.

A second mode of the present invention relates to a method for obtaininginformation of a test substance. A method for obtaining information of atest substance according to the present mode includes: causing (S12,S21) a magnetic particle (21) to bind to a test substance (11) in aspecimen (10); selectively collecting (S14, S15, S23, S24) at least thetest substance (11) from the specimen (10) by use of the magneticparticle (21) bound to the test substance (11); immobilizing (S16, S26)the test substance (11) collected from the specimen (10), onto a baseplate (80); and obtaining (S17, S27) information regarding a structureof the test substance (11) from the test substance (11) immobilized onthe base plate (80).

In the method for obtaining the information of the test substanceaccording to the present mode, the test substance is selectivelycollected from the specimen by means of a magnet, for example, and thecollected test substance is immobilized on the base plate. At this time,impurities other than the test substance contained in the specimen areseparated from the test substance and are suppressed from beingtransferred to the base plate. Thus, to the base plate, substantiallyonly the test substance is transferred and immobilized. Thus, theinformation regarding the structure of the test substance can beaccurately obtained.

A third mode of the present invention relates to a method for obtaininginformation of a test substance. A method for obtaining information of atest substance according to the present mode includes forming (S11 toS13, S21, S22) a complex (60, 61) by causing a capture substance (40)including a fluorescent dye (41) to bind to a test substance (11) in aspecimen (10); selectively collecting (S14, S15, S23, S24) at least thecomplex (60, 61) from the specimen (10); immobilizing (S16, S26) thecomplex (60, 61) collected from the specimen (10), onto a base plate(80); and obtaining (S17, S27) information regarding a structure of thetest substance (11) on the basis of bright spots corresponding tofluorescence emitted from a plurality of fluorescent dyes (41) eachbound to the complex (60) immobilized on the base plate (80).

In the method for obtaining the information of the test substanceaccording to the present mode, the complex is selectively collected fromthe specimen and the collected complex is immobilized on the base plate.At this time, substances other than the test substance contained in thespecimen and impurities such as a capture substance that has not formedthe complex are separated from the complex and are suppressed from beingtransferred to the base plate. Thus, to the base plate, substantiallyonly the test substance is transferred and immobilized. Thus, theinformation regarding the structure of the test substance can beaccurately obtained on the basis of the bright spots corresponding tofluorescence emitted from the fluorescent dyes.

A fourth mode of the present invention relates to a method for obtaininginformation of a test substance. A method for obtaining information of atest substance according to the present mode includes: separating (S14,S23) a test substance (11) and an impurity (13) in a specimen (10) fromeach other; immobilizing (S16, S26) the test substance (11) separatedfrom the impurity (13), onto a base plate (80); and obtaining (S17, S27)information regarding a structure of the test substance (11) immobilizedon the base plate (80).

In the method for obtaining the information of the test substanceaccording to the present mode, the test substance and the impurity areseparated from each other, and the separated test substance isimmobilized on the base plate. At this time, substances other than thetest substance contained in the specimen and impurities such as acapture substance used in the separation are separated from the testsubstance and are suppressed from being transferred to the base plate.Thus, to the base plate, substantially only the test substance istransferred and immobilized. Thus, the information regarding thestructure of the test substance can be accurately obtained.

In the method for obtaining the information of the test substanceaccording to the present mode, the test substance (11) is a protein as atest target contained in the specimen (10), and the impurity (13)includes a protein other than the protein as the test target.

In the method for obtaining the information of the test substanceaccording to the present mode, the specimen (10) is cerebrospinal fluid.

According to the present invention, information regarding the structureof a test substance can be accurately obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing an information obtaining method accordingto Embodiment 1;

FIG. 2A is a schematic diagram showing a binding step, a separationpreparing step, and a labeling step according to Embodiment 1;

FIG. 2B is a schematic diagram showing a separating step according toEmbodiment 1;

FIG. 3A is a schematic diagram showing a detaching step according toEmbodiment 1;

FIG. 3B is a schematic diagram showing the detaching step according toEmbodiment 1;

FIG. 4 is a schematic diagram showing an immobilizing step according toEmbodiment 1;

FIG. 5 is a flow chart showing an information obtaining method accordingto Embodiment 2;

FIG. 6 is a schematic diagram showing an immobilizing step according toEmbodiment 2;

FIG. 7 is a flow chart showing an information obtaining method accordingto Embodiment 3;

FIG. 8A is a schematic diagram showing a separation preparing step and alabeling step according to Embodiment 3;

FIG. 8B is a schematic diagram showing a separating step according toEmbodiment 3;

FIG. 9A is a schematic diagram showing a detaching step according toEmbodiment 3;

FIG. 9B is a schematic diagram showing the detaching step according toEmbodiment 3;

FIG. 9C is a schematic diagram showing a binding step according toEmbodiment 3;

FIG. 10 is a flow chart showing an information obtaining methodaccording to Embodiment 4;

FIG. 11 is a schematic diagram showing a binding step according toEmbodiment 4;

FIG. 12 is a flow chart showing an information obtaining step accordingto Embodiments 1 to 4;

FIG. 13A is a schematic diagram showing that all fluorescent dyes are ina light emitting state in the information obtaining step according toEmbodiments 1 to 4;

FIG. 13B is a schematic diagram showing that some of the fluorescentdyes are in the light emitting state in the information obtaining stepaccording to Embodiments 1 to 4;

FIG. 13C is a schematic diagram showing that some of the fluorescentdyes are in the light emitting state in the information obtaining stepaccording to Embodiments 1 to 4;

FIG. 14 is a diagram describing the procedure of obtaining asuper-resolution image and classifying bright spots into a group in theinformation obtaining step according to Embodiments 1 to 4;

FIG. 15 is a diagram showing an example of a screen displayed on adisplay unit in the information obtaining step according to Embodiments1 to 4;

FIG. 16A shows super-resolution images obtained through the procedure ofverification of Embodiment 1;

FIG. 16B shows fluorescence images obtained through the procedure ofverification of Embodiment 1;

FIG. 16C is a graph showing the relationship between the number ofbright spots on the fluorescence images obtained in the verification ofEmbodiment 1 and the concentration of the test substance;

FIG. 17A shows a super-resolution image obtained through the procedureof verification of Comparative Example;

FIG. 17B shows fluorescence images obtained through the procedure ofverification of Comparative Example;

FIG. 17C is a graph showing the relationship between the number ofbright spots on the fluorescence images obtained in the verification ofComparative Example and the concentration of the test substance;

FIG. 18 is a schematic diagram showing a configuration of a detectionapparatus for automatically performing the information obtaining stepaccording to Embodiments 1 to 4;

FIG. 19A is a schematic diagram for describing a configuration ofrelated art; and

FIG. 19B is a schematic diagram for describing the configuration of therelated art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments 1 to 4 shown below indicate embodiments of the presentdisclosure. A specimen is a liquid taken from a living body and is CSF(cerebrospinal fluid), blood, plasma, interstitial fluid, urine, or thelike, for example. A test substance is a cell, a polypeptide, a protein,a nucleic acid, an exosome, a carbohydrate chain, or the like, and inthe information obtaining method of Embodiments 1 to 4, a multimer isappropriate. The test substance is an amyloid β oligomer which is madeup of polymerized amyloid β monomers, a tau oligomer which is made up ofpolymerized tau proteins, or the like, for example. In Embodiments 1 to4, the specimen is CSF, and the test substance is amyloid β.

Embodiment 1

As shown in FIG. 1, an information obtaining method of Embodiment 1includes process steps of steps S11 to S17. Steps S11 to S16 may bemanually performed by an operator or may be automatically performed by aprocessing apparatus. The information obtaining step of step S17 may beperformed by the operator by use of a microscope, or may beautomatically performed by a detection apparatus. The detectionapparatus which automatically performs the information obtaining step ofstep S17 will be described later with reference to FIG. 18.

As shown in FIG. 2A, a specimen 10 contains a test substance 11 and animpurity 12. The impurity 12 is a substance which is other than the testsubstance 11 and which is not necessary. The impurity 12 is a proteinother than the test substance 11, for example. In each step shown inFIG. 1, a solid phase 20, a second capture substance 30, a third capturesubstance 40, and a first capture substance 50 are used. In thefollowing, each step shown in FIG. 1 is described with reference to FIG.2A to FIG. 4. In a forming step composed of steps S11 to S13, steps S11to S13 are simultaneously performed. In the forming step, each capturesubstance is bound to the test substance 11 in the specimen 10, wherebya complex 60 is formed. In a collecting step composed of steps S14 andS15, the complex 60 is selectively collected from the specimen 10.

In the binding step of step S11, the first capture substance 50 is boundto the test substance 11. As shown in FIG. 2A, the first capturesubstance 50 is a substance capable of binding to a base plate 80 shownin FIG. 4, and includes a binding substance 51 and an antibody 52 havingthe binding substance 51 bound thereto. The binding substance 51specifically binds to a binding partner 81 of the base plate 80described later, and the antibody 52 specifically binds to the testsubstance 11. As a result of the antibody 52 binding to the testsubstance 11, the first capture substance 50 and the test substance 11are bound to each other.

In a separation preparing step of step S12, the solid phase 20 is boundto the complex 60 through the second capture substance 30. As shown inFIG. 2A, the solid phase 20 includes a magnetic particle 21 and a secondbinding partner 22 having the magnetic particle 21 bound thereto. Thesecond capture substance 30 includes a second binding substance 31 andan antibody 32 having the second binding substance 31 bound thereto. Thesecond binding partner 22 specifically binds to the second bindingsubstance 31 and the antibody 32 specifically binds to the testsubstance 11. The second binding partner 22 and the second bindingsubstance 31 are bound to each other and the antibody 32 and the testsubstance 11 are bound to each other, whereby the solid phase 20 and thecomplex 60 are bound to each other.

As described above, if the specific binding of the complex 60 to thesolid phase 20 is performed through the second capture substance 30capable of binding to the solid phase 20, the complex 60 can be detachedfrom the solid phase 20 in a detaching step in the latter stage. Inaddition, the solid phase 20 and the test substance 11 can be stablybound to each other.

A combination of the second binding substance 31 and the second bindingpartner 22 is selected from combinations of: an antigen and an antibodythereto; a ligand and a receptor therefor; an oligonucleotide and acomplementary strand thereof; and biotins including biotin and biotinanalogs such as desthiobiotin and avidins including avidin and avidinanalogs such as streptavidin. Then, the second binding substance 31 andthe second binding partner 22 can be stably bound to each other.Examples of the combination of a ligand and a receptor therefor includecombinations of: an enzyme and a substrate therefor; and a signalsubstance such as a hormone or a neurotransmitter and a receptortherefor. Alternatively, a combination in which the second bindingsubstance 31 is an anti- hapten and the second binding partner 22 is ananti-hapten antibody may be employed. Also in this case, the secondbinding substance 31 and the second binding partner 22 can be stablybound to each other.

In Embodiment 1, the second binding substance 31 is a dinitrophenylgroup and the second binding partner 22 is an anti-dinitrophenyl groupantibody. In this case, if a dinitrophenyl amino acid is used as areleaser described later, the second binding substance 31 and the secondbinding partner 22 bound to each other can be easily detached from eachother.

In Embodiment 1, the first capture substance 50 and the second capturesubstance 30 are configured to be different from each other, but theconfiguration is not limited thereto. For example, instead of the firstcapture substance 50 and the second capture substance 30, the bindingsubstance 51 and the second binding substance 31 are bound to a singleantibody, whereby another capture substance may be configured. However,in this case, in a separating step, not the test substance 11 alone butthe other capture substance that does not have the test substance 11bound thereto is also taken out together with the test substance 11.Thus, in an immobilizing step, the other capture substance that does nothave the test substance 11 bound thereto is immobilized to the bindingpartner 81 of the base plate 80. Thus, the test substance 11 becomesless likely to be bound to the binding partner 81, and the efficiency ofimmobilizing the test substance 11 to the base plate 80 is decreased.Therefore, in order to smoothly perform immobilization of the testsubstance 11 onto the base plate 80, it is preferable that the firstcapture substance 50 and the second capture substance 30 are configuredto be different from each other and individually bound to the testsubstance 11.

In a labeling step of step S13, the third capture substance 40 is boundto the test substance 11. As shown in FIG. 2A, the third capturesubstance 40 includes a fluorescent dye 41 which is a fluorescent label,and an antibody 42 labeled with the fluorescent dye 41. The antibody 42specifically binds to the test substance 11. As a result of the antibody42 and the test substance 11 binding to each other, a fluorescent labelis provided to the test substance 11.

In Embodiment 1, at least portions of the epitopes of the antibody 52 ofthe first capture substance 50, the antibody 32 of the second capturesubstance 30, and the antibody 42 of the third capture substance 40overlap one another, and steps S11 to S13 are simultaneously performed.In such a case where at least portions of the epitopes of the respectiveantibodies overlap one another, it is preferable that steps S11 to S13are performed simultaneously. More specifically, a step of mixing thefirst capture substance 50 into the specimen 10 in step S11, a step ofmixing the second capture substance 30 into the specimen 10 in step S12,and a step of mixing the third capture substance 40 into the specimen 10in step S13 are preferably performed simultaneously.

For example, if the second capture substance 30 is mixed into thespecimen 10 before the first capture substance 50 is mixed into thespecimen 10, the binding site in the test substance 11 is occupied bythe second capture substance 30, which could hinder smooth binding ofthe first capture substance 50 to the test substance 11. If the mixingtimings are different in such a manner, the substances mixed later areless likely to bind to the test substance 11. By simultaneously mixingthe first capture substance 50, the second capture substance 30, and thethird capture substance 40 into the specimen 10, each substance isallowed to smoothly bind to the test substance 11.

When the epitopes of the respective antibodies do not overlap oneanother, it is possible that steps S11 to S13 are not simultaneouslyperformed, and the order of steps S11 to S13 is also not limited to theorder shown in FIG. 1.

At the time point when steps S11 to S13 have ended, as shown in FIG. 2A,the first capture substance 50, the second capture substance 30, and thethird capture substance 40 are bound to the test substance 11 in thespecimen 10 in the container, to form the complex 60, whereby a state inwhich the solid phase 20 is bound to the complex 60 is established.

In the separating step of step S14, the solid phase 20 is selectivelyseparated, whereby the test substance 11 is separated from the specimen10. Specifically, by the complex 60 being separated from impurities 13,the test substance 11 is taken out from the specimen 10. The impurities13 of Embodiment 1 include: the impurity 12; and the capture substances30, 40, and 50 which have not formed the complex 60, as shown in FIG.2B.

As shown in FIG. 2B, the separation of the complex 60 from theimpurities 13 in the separating step is performed by use of a magnet 70,for example. When the magnet 70 is brought close to the container, themagnetic particle 21 is attracted by the magnetic force to the innerwall of the container at which the magnet 70 is located. At this time,since the magnetic particle 21 and the test substance 11 are integratedwith each other, the test substance 11 is also attracted to the innerwall of the container, together with the magnetic particle 21. In thisstate, the liquid in the container is removed. The removed liquidcontains the impurities 13. Thus, the test substance 11 and theimpurities 13 are separated from each other, and the test substance 11is taken out from the specimen 10.

In Embodiment 1, before the separating step, the step of causing thefirst capture substance 50 to bind to the test substance 11 isperformed. Thus, in addition to the separation of the impurity 12, thefirst capture substance 50 that has not bound to the test substance 11can be separated from the test substance 11, in the separating step.Accordingly, the first capture substance 50 that has not bound to thetest substance 11 can be suppressed from being transferred to the baseplate 80 in the immobilizing step of step S16. Similarly, before theseparating step, the steps of causing the second capture substance 30and the third capture substance 40 to bind to the test substance 11 areperformed. Thus, in addition to the separation of the impurity 12, thesecond capture substance 30 and the third capture substance 40 that havenot bound to the test substance 11 can be separated from the testsubstance 11 in the separating step. Accordingly, the second capturesubstance 30 and the third capture substance 40 that have not bound tothe test substance 11 can be suppressed from being transferred to thebase plate 80 in the immobilizing step of step S16.

It should be noted that, in a case where the step of causing the firstcapture substance 50 to bind to the test substance 11 is performed afterthe separating step, it is preferable to further perform a step ofremoving the first capture substance 50 that has not bound to the testsubstance 11. The “separation in the separating step” is not limited toseparation of the complex 60 only, but is a concept that encompassesseparation of the complex 60 with substances other than the complex 60slightly included.

In Embodiment 1, on the basis of a difference in immunoreaction, i.e.,on the basis of the specific binding between the antibody 32 of thesecond capture substance 30 and the test substance 11 throughantigen-antibody reaction, the solid phase 20 is selectively collectedin the separating step, whereby the test substance 11 and the impurities13 are separated from each other. However, not limited thereto, themethod for separating the test substance 11 and the impurity 12 fromeach other may be any separating method based on at least one of adifference in specific gravity, a difference in size, a difference inelectrical property, and a difference in immunoreaction between thecomplex 60 and the impurities 13. Specifically, the separating methodmay be gel filtration, electrophoresis, immunoreaction, or the like.Then, the separation can be performed appropriately. If a difference inimmunoreaction is used as described above, the binding specificitybetween the complex 60 and the solid phase 20 is enhanced, and thus, thetest substance 11 can be more accurately separated from the impurities13.

The separation of the test substance 11 is preferably performed by useof the solid phase 20 as described above. The solid phase 20 preferablyincludes the magnetic particle 21 as described above, and preferably,the magnetic particle 21 bound to the test substance 11 is attracted bymeans of the magnet 70, to separate the test substance 11. By attractingthe magnetic particle 21 by means of the magnet 70, the separationbetween the test substance 11 and the impurities 13 can be smoothlyperformed.

In the detaching step of step S15, the complex 60 is detached from thesolid phase 20. Specifically, as shown in FIG. 3A, the releaser is mixedinto the liquid containing the separated complex 60, whereby the solidphase 20 is detached from the complex 60. Accordingly, the solid phase20 is released in the liquid. Then, as shown in FIG. 3B, the magnet 70is brought close to the container, and the solid phase 20 is attractedby means of the magnet 70 to the inner wall of the container at whichthe magnet 70 is located. In this state, the liquid in the container istaken out. The liquid that has been taken out contains the complex 60.In this manner, the complex 60 is detached from the solid phase 20.Since the solid phase 20 is detached, occurrence of noise caused by thesolid phase 20 bound to the test substance 11, other substancesnonspecifically bound to the solid phase 20, and the like is suppressedin observation of the test substance 11.

The detachment between the complex 60 and the solid phase 20 may berealized on the basis of immunological separation due to competitivereaction, or may be realized on the basis of chemical separation by useof a reducing agent, for example. In Embodiment 1, as shown in FIG. 3A,as a result of the second binding partner 22 and the second bindingsubstance 31 being detached from each other, the complex 60 and thesolid phase 20 are detached from each other. However, the detachmentbetween the complex 60 and the solid phase 20 is not limited thereto,and it is sufficient that, at any portion that connects the magneticparticle 21 and the complex 60 to each other, the complex 60 side andthe solid phase 20 side are detached from each other.

In the immobilizing step of step S16, as shown in FIG. 4, the firstcapture substance 50 is bound to the base plate 80, whereby the testsubstance 11 is immobilized on the base plate 80. The base plate 80 isformed as a glass plate or the like, for example. The base plate 80includes the binding partner 81 which specifically binds to the bindingsubstance 51 of the first capture substance 50. In the immobilizingstep, through specific binding between the binding partner 81 and thebinding substance 51, the test substance 11 is immobilized on the baseplate 80. Thus, if the test substance 11 is specifically bound to thebase plate 80 not through physical adsorption but through mediation bythe first capture substance 50, the test substance 11 can be stablyimmobilized on the base plate 80 while transfer of the impurities 13 tothe base plate 80 is further suppressed.

The combination of the binding substance 51 and the binding partner 81is selected from combinations of: an antigen and an antibody thereto; aligand and a receptor therefor; an oligonucleotide and a complementarystrand thereof; and biotins including biotin and biotin analogs such asdesthiobiotin and avidins including avidin and avidin analogs such asstreptavidin. Then, the binding substance 51 and the binding partner 81can be stably bound to each other. Examples of the combination of aligand and a receptor therefor include combinations of: an enzyme and asubstrate therefor; a signal substance such as a hormone or aneurotransmitter, and a receptor therefor. In Embodiment 1, the bindingsubstance 51 is a type of the biotins, and the binding partner 81 is atype of the avidins. In this case, due to high affinity between thebinding substance 51 and the binding partner 81, the test substance 11can be more stably immobilized on the base plate 80.

In Embodiment 1, the labeling step is performed before the immobilizingstep. Thus, direct attachment of the third capture substance 40 to thebase plate 80 is suppressed, and information regarding the structure ofthe test substance 11 can be accurately obtained in an informationobtaining step described later. In addition, after the complex 60 isdetached from the solid phase 20, the first capture substance 50 boundto the complex 60 which has been detached from the solid phase 20 isbound to the base plate 80, whereby the test substance 11 is immobilizedon the base plate 80. Therefore, since the solid phase 20 is nottransferred to the base plate 80, information regarding the structure ofthe test substance 11 can be accurately obtained.

In the information obtaining step of step S17, information regarding thestructure of the test substance 11 is obtained from the test substance11 immobilized on the base plate 80. It should be noted that“information regarding the structure of the test substance” is a conceptthat broadly encompasses the size, the morphology, the structure, thechemical bond, the aggregation degree, and the like of the testsubstance 11. Details of the information obtaining step are describedlater with reference to FIG. 12.

In Embodiment 1, the test substance 11 is separated from the specimen10, and the separated test substance 11 is immobilized on the base plate80. Thus, the impurities 13 are suppressed from being transferred to thebase plate 80, and substantially only the test substance 11 istransferred to the base plate 80 and immobilized thereon. Therefore,information regarding the structure of the test substance 11 can beaccurately obtained. Since the impurities 13 are suppressed from beingtransferred to the base plate 80, even when the concentration of thetest substance 11 is low, a signal derived from the test substance 11 isless likely to be obstructed by signals derived from the impurities 13.Accordingly, information regarding the structure of the test substance11 can be highly accurately obtained while influence of the impurities13 is suppressed.

Information regarding the structure of the test substance 11 changesdepending on the disease progression and the like, and thus, is usefulin pathological diagnosis, determination of an administration policy,and the like. For example, when the test substance 11 is amyloid β,information regarding the structure of amyloid β can be helpful indiagnosis of Alzheimer's disease and determination of an administrationpolicy therefor. According to Embodiment 1, since the impurities 13 areremoved, even when the concentration of amyloid β is low, informationregarding the structure of amyloid β can be highly accurately obtained.Thus, even in a case where the concentration of amyloid β is low such asduring the early stages of Alzheimer's disease, the obtained informationregarding the structure can be helpful in diagnosis of diseaseprogression.

Embodiment 2

As shown in FIG. 5, in Embodiment 2, compared with Embodiment 1, thedetaching step of step S15 is omitted from the flow chart shown inFIG. 1. That is, in Embodiment 2, after steps S11 to S13 are performedas in Embodiment 1, steps S16 and S17 are performed, without thedetaching step being performed. The collecting step of Embodiment 2 isconfigured as the separating step of step S14.

In Embodiment 2, as a result of steps S11 to S14 being performed, thetest substance 11 and the impurities 13 are separated from each otherand the test substance 11 is taken out from the specimen 10, as inEmbodiment 1 and as shown in FIG. 2B. Subsequently, in Embodiment 2, theimmobilizing step of step S16 is performed. In the immobilizing step ofstep S16, as shown in FIG. 6, the first capture substance 50 is bound tothe base plate 80, whereby the test substance 11 is immobilized on thebase plate 80. At this time, different from Embodiment 1, the testsubstance 11 has the solid phase 20 bound thereto. Then, in theinformation obtaining step of step S17, as in Embodiment 1, informationregarding the structure of the test substance 11 is obtained from thetest substance 11 immobilized on the base plate 80.

Also in Embodiment 2, as in Embodiment 1, the impurities 13 aresuppressed from being transferred to the base plate 80, andsubstantially only the test substance 11 is transferred to the baseplate 80 and immobilized thereon. Thus, information regarding thestructure of the test substance 11 can be accurately obtained. InEmbodiment 2, since the step for removing the solid phase 20 is omitted,the test substance 11 is not removed in the step for removing the solidphase 20. It should be noted that in a case where the detaching step isomitted, the magnetic particle 21 may be bound to the test substance 11not through the second capture substance 30. For example, an antibodybound to the magnetic particle is bound to the test substance 11,whereby the magnetic particle 21 may be bound to the test substance 11.Alternatively, the magnetic particle 21 may be directly bound to thetest substance 11.

Embodiment 3

As shown in FIG. 7, in Embodiment 3, compared with Embodiment 1, thebinding step is performed between the detaching step and theimmobilizing step in the flow chart shown in FIG. 1. That is, inEmbodiment 3, after the separation preparing step, the labeling step,the separating step, and the detaching step are performed, the bindingstep is performed. In the forming step composed of steps S21 and S22,steps S21 and S22 are simultaneously performed. In the forming step,each capture substance is bound to the test substance 11 in the specimen10, to form a complex 61. In the collecting step composed of steps S23and S24, the complex 61 is selectively collected from the specimen 10.

In the separation preparing step of step S21, as shown in FIG. 8A, thesolid phase 20 is bound to the test substance 11 through the secondcapture substance 30. Binding between the solid phase 20 and the testsubstance 11 is the same as that in Embodiment 1. In the labeling stepof step S22, as shown in FIG. 8A, the third capture substance 40 isbound to the test substance 11. Binding between the third capturesubstance 40 and the test substance 11 is the same as that in Embodiment1.

In FIG. 7, steps S21 and S22 are arranged in this order for convenience,but in actuality, steps S21 and S22 are simultaneously performed. Itshould be noted that it is possible that steps S21 and S22 are notsimultaneously performed, and the order of steps S21 and S22 may be thereverse of the order shown in FIG. 7. However, in order to cause thesecond capture substance 30 and the third capture substance 40 tosmoothly bind to the test substance 11, it is preferable that steps S21and S22 are simultaneously performed.

At the time point when steps S21 and S22 have ended, as shown in FIG.8A, in the specimen 10 in the container, the second capture substance 30and the third capture substance 40 are bound to the test substance 11,to form the complex 61, whereby a state in which the solid phase 20 isbound to the complex 61 is established.

In the separating step of step S23, the solid phase 20 is selectivelyseparated, whereby the test substance 11 is separated from the specimen10. Specifically, by the complex 61 being separated from the impurities13, the test substance 11 is taken out from the specimen 10. Theimpurities 13 of Embodiment 3 include: the impurity 12; and the capturesubstances 30 and 40 which have not formed the complex 61, as shown inFIG. 8B. As shown in FIG. 8B, the separation of the test substance 11from the impurities 13 in the separating step is performed by use of themagnet 70, for example, as in Embodiment 1.

In the detaching step of step S24, the complex 61 is detached from thesolid phase 20. Specifically, as shown in FIG. 9A, a releaser is mixedinto the liquid containing the separated complex 61, whereby the solidphase 20 is detached from the complex 61. Then, as shown in FIG. 9B, thesolid phase 20 is attracted by means of the magnet 70 to the inner wallof the container at which the magnet 70 is located. In this state, theliquid in the container is taken out. The liquid that has been taken outcontains the complex 61. In this manner, the complex 61 is detached fromthe solid phase 20. Since the solid phase 20 is detached, occurrence ofnoise caused by the solid phase 20 bound to the test substance 11, othersubstances nonspecifically bound to the solid phase 20, and the like canbe suppressed in observation of the test substance 11.

In the binding step of step S25, the first capture substance 50 is boundto the test substance 11. As shown in FIG. 9C, the first capturesubstance 50 is mixed into the liquid containing the test substance 11taken out in the detaching step. As a result of the antibody 52 and thetest substance 11 binding to each other, the first capture substance 50and the test substance 11 are bound to each other, whereby the complex60 is generated. It should be noted that the binding step may beperformed between the separating step and the detaching step.

In the immobilizing step of step S26, as in Embodiment 1, the firstcapture substance 50 is bound to the base plate 80, whereby the testsubstance 11 is immobilized on the base plate 80 as shown in FIG. 4.

Also in Embodiment 3, as in Embodiment 1, the impurities 13 aresuppressed from being transferred to the base plate 80, andsubstantially only the test substance 11 is transferred to the baseplate 80 and immobilized thereon. Thus, information regarding thestructure of the test substance 11 can be accurately obtained. After thecomplex 61 is detached from the solid phase 20, the first capturesubstance 50 is bound to the test substance 11 detached from the solidphase 20, and the test substance 11 is immobilized on the base plate 80through the first capture substance 50. Therefore, since the solid phase20 is not transferred to the base plate 80, information regarding thestructure of the test substance 11 can be accurately obtained.

Embodiment 4

As shown in FIG. 10, in Embodiment 4, compared with Embodiment 3, thedetaching step of step S24 is omitted from the flow chart shown in FIG.7. That is, in Embodiment 4, as in Embodiment 3, after steps S21 to S23are performed, steps S25 to S27 are performed, without the detachingstep being performed. The collecting step of Embodiment 4 is configuredas the separating step of step S23.

In Embodiment 4, as a result of steps S21 to S23 being performed, thetest substance 11 and the impurities 13 are separated from each other,whereby the test substance 11 is taken out from the specimen 10, as inEmbodiment 3 and as shown in FIG. 8B. Subsequently, in Embodiment 4, thebinding step of step S25 is performed. In the binding step of step S25,as shown in FIG. 11, the first capture substance 50 is mixed into theliquid containing the complex 61 separated in the separating step. As aresult of the antibody 52 and the test substance 11 binding to eachother, the first capture substance 50 and the test substance 11 arebound to each other, whereby the complex 60 is generated. In theimmobilizing step of step S26, as shown in FIG. 6, the first capturesubstance 50 is bound to the base plate 80, whereby the test substance11 is immobilized on the base plate 80. Then, in the informationobtaining step of step S27, as in Embodiment 3, information regardingthe structure of the test substance 11 is obtained from the testsubstance 11 immobilized on the base plate 80.

Also in Embodiment 4, as in Embodiment 3, the impurities 13 aresuppressed from being transferred to the base plate 80, andsubstantially only the test substance 11 is transferred to the baseplate 80 and immobilized thereon. Thus, information regarding thestructure of the test substance 11 can be accurately obtained. Inaddition, in Embodiment 4, since the step for removing the solid phase20 is omitted, the test substance 11 is not removed in the step forremoving the solid phase 20.

<Information Obtaining Step>

Next, details of the information obtaining step of Embodiments 1 to 4are described.

The information obtaining step includes a step of measuring the testsubstance 11 on the base plate 80 by means of a super-resolutionfluorescence microscope having a resolution exceeding the diffractionlimit of light. When a microscope having a resolution exceeding thediffraction limit of light is used, information regarding the structureof the test substance 11 can be obtained at a resolution exceeding thediffraction limit of light. It should be noted that the informationobtaining step may be automatically performed by a detection apparatus100 described later with reference to FIG. 18.

FIG. 12 is a flow chart showing the information obtaining step. In thedescription below, schematic diagrams shown in FIG. 13A to FIG. 13C arereferenced as appropriate. FIG. 13A to FIG. 13C show the test substance11 immobilized on the base plate 80.

Here, the fluorescent dye 41 is configured to be switched between alight emitting state in which the fluorescent dye 41 generatesfluorescence and a quenched state in which the fluorescent dye 41 doesnot generate fluorescence, when excitation light is continually appliedto the fluorescent dye 41. As the fluorescent dye 41, a commerciallyavailable dye can be used. In FIG. 13A to FIG. 13C, the fluorescent dye41 in the light emitting state is indicated by a black circle, and thefluorescent dye 41 in the quenched state is indicated by a while circle.

As shown in FIG. 12, in step S101, the excitation light is applied tothe fluorescent dyes 41 on the base plate 80. As shown in FIG. 13A, inthe initial state, all of the fluorescent dyes 41 are in the lightemitting state. When the excitation light starts to be applied in thisstate, fluorescence is excited from all of the fluorescent dyes 41.Then, when the excitation light is continually applied to thefluorescent dyes 41, the distribution of the fluorescent dyes 41 in thelight emitting state changes as shown in, for example, FIG. 13B and FIG.13C, with the lapse of time.

In step S102, while the excitation light is applied to the fluorescentdyes 41, image capturing of the generated fluorescence is performed, andimages of the fluorescent dyes 41 are obtained. In step S102, the imagecapturing is repeated while the excitation light is being applied to thefluorescent dyes 41, and 3000 images are obtained, for example. Sincethe distribution of the fluorescent dyes 41 in the light emitting statechanges in accordance with the lapse of time as described above, thedistribution of fluorescence on the obtained images is different for therespective timings of the image capturing.

In step S103, whether a predetermined time period has elapsed andobtaining of necessary images has ended is determined. When obtaining ofnecessary images has been completed, the process is advanced to stepS104. When the images are obtained in this manner, information regardingthe structure of the test substance 11 can be obtained in a step of thelatter stage.

The images may be obtained by steps based on the technique according toSTORM, PALM, STED, or SIM, instead of steps S101 to S103. In a casewhere the images are obtained by the steps based on STORM, thefluorescent dye 41 is configured to be switched between an active statein which the fluorescent dye 41 generates fluorescence and an inactivestate in which the fluorescent dye 41 does not generate fluorescence.Then, by the fluorescent dye 41 being switched between the active stateand the inactive state by two kinds light, a plurality of images havingdifferent distribution of fluorescence are obtained, similarly to theabove.

Subsequently, in step S104, a super-resolution image is created.

As shown in FIG. 14, a super-resolution image is created on the basis ofa plurality of fluorescence images obtained in step S102 shown in FIG.12. Specifically, for each fluorescence image, bright spots offluorescence are extracted through Gauss fitting. Accordingly, on atwo-dimensional plane, coordinates of each bright spot are obtained.Here, as a result of Gauss fitting, as to a bright spot of afluorescence region that matches with a reference waveform in apredetermined range, a bright spot region of a size corresponding tothis range is assigned. As to a bright spot of a fluorescence regionthat matches, at one point, with the reference waveform, a bright spotregion of a lowest-level size is assigned. The bright spot regionsobtained from each of the fluorescence images are superposed, whereby asuper-resolution image is created.

Thus, in a case where 3000 fluorescence images are obtained in stepS102, bright spots are extracted from the 3000 fluorescence images andthe bright spot regions of the extracted bright spots are superposed,whereby a super-resolution image of the fluorescence images is created.

With reference back to FIG. 12, in step S105, information regarding thestructure of the test substance 11 is obtained. In step S105, asinformation regarding the structure of the test substance 11, the size,the morphology, the structure, the aggregation degree, and the likeregarding the test substance 11 are obtained.

In step S105, information regarding the structure of the test substance11 is obtained in the following procedure.

As shown in FIG. 14, bright spots extracted at the creation of thesuper-resolution image obtained in step S104 shown in FIG. 12 areclassified into a group corresponding to aggregated test substance 11.That is, all the bright spots extracted from the plurality offluorescence images are mapped on a coordinate plane. Subsequently, thecoordinate plane is scanned for a reference region of a predeterminedsize, and the number of bright spots contained in the reference regionis obtained. Then, the position of a reference region in which thenumber of bright spots contained therein is greater than a threshold andgreater than in the surrounding area is extracted, and the bright spotscontained in the reference region at the extracted position areclassified into one group. The one group thus obtained is regarded asone aggregate of the test substance 11.

The method for classifying bright spots into one group is not limitedthereto, and another clustering technique may be employed. For example,a region that has a brightness not less than a predetermined thresholdon a fluorescence image generated by totaling all fluorescence imagesmay be regarded as one aggregate. Alternatively, a region that has abrightness not less than a predetermined threshold on a fluorescenceimage obtained by performing image capturing of fluorescence excitedfrom all the fluorescent dyes 41 immediately after the start of stepS101 in the information obtaining step may be regarded as one aggregate.

Subsequently, for each aggregate of the test substance 11, the followinginformation is obtained on the basis of the super-resolution image. Thatis, as the size of the test substance 11, the length in the longitudinaldirection, the length in the short direction, the perimeter, the area,and the like are obtained. As the morphology of the test substance 11,the aspect ratio, the circularity, the number of branches, the anglebetween branches, and the like are obtained. The aspect ratio isobtained by dividing the length in the longitudinal direction by thelength in the short direction, for example. As the structure of the testsubstance 11, which among the primary structure, the secondarystructure, the tertiary structure, and the quaternary structure ofprotein corresponds to the aggregate of the test substance 11 isobtained. As the aggregation degree of the test substance 11, the numberof monomers forming the aggregate is obtained. The number of monomers isobtained by comparing the standard size of a monomer with the size ofthe aggregate.

In step S105, information regarding the structure of the test substance11 is obtained on the basis of the super-resolution image. However, notlimited thereto, information regarding the structure of the testsubstance 11 may be obtained on the basis of a fluorescence imageobtained by performing image capturing of the fluorescence generatedfrom the fluorescent dyes 41. For example, information regarding thestructure of the test substance 11 may be obtained on the basis of afluorescence image obtained by performing image capturing of thefluorescence generated from all the fluorescent dyes 41 immediatelyafter the start in step S101. However, in this case, analysis cannot beperformed at a resolution exceeding the diffraction limit of light.Therefore, it is preferable that information regarding the structure ofthe test substance 11 is obtained on the basis of a super-resolutionimage as described above.

With reference back to FIG. 12, in step S106, information obtained instep S105 is outputted. Specifically, the obtained information isdisplayed on a display unit implemented as a display. Other than this,the obtained information may be outputted as a sound from a speaker, ormay be transmitted as digital data to another apparatus.

FIG. 15 is a schematic diagram showing a screen 90 displayed on thedisplay unit in step S106.

The screen 90 includes images 91, 92 and a region 93. The image 91 isthe super-resolution image obtained in step S104 shown in FIG. 12. Theimage 92 is an enlarged image of a portion of the image 91. The region93 is a region in which to display the information regarding thestructure of the test substance 11 obtained in step S105 shown in FIG.12. When the screen 90 as shown in FIG. 15 is displayed in theinformation obtaining step, a doctor and the like, for example, canvisually understand the super-resolution image and the informationregarding the structure of the test substance 11, and thus, can smoothlydiagnose the disease condition and determine an administration policy.

Verification of Embodiment 1

Next, the verification of Embodiment 1 performed by the inventors isdescribed. In this verification, the inventors obtained super-resolutionimages in accordance with the procedure of Embodiment 1, and obtainedsuper-resolution images in accordance with the procedure of ComparativeExample in which impurities were not removed.

[Preparation of Specimen]

Amyloid β peptide 1-42 human (manufactured by Eisai) was diluted withCSF (manufactured by Access Biologicals), to prepare a plurality ofspecimens each containing a test substance and having differentconcentrations. The prepared specimens correspond to the specimen 10 ofEmbodiment 1.

[Preparation of Base Plate]

By the following method, glass base plates each modified withstreptavidin were prepared. The prepared glass base plates correspond tothe base plate 80 of Embodiment 1. (1) A through-hole having a 6 mmdiameter was made in a silicone rubber sheet (TIGERS POLYMERCORPORATION, SR-50) and the silicone rubber sheet was attached to an MAScoated glass (manufactured by Matsunami Glass Ind., Ltd.). (2) 0.5 μL of30 μg/mL biotin-bound BSA was dropped on the glass inside the siliconerubber sheet, and the resultant object was left to stand for one hour atroom temperature. (3) By use of 20 μL of an HISCL washing liquid(manufactured by Sysmex Corporation), washing by pipetting was performedthree times in total. (4) 20 μL of a 1%BSA/0.05%PBST solution wasdropped and the resultant object was left to stand overnight at 4° C.(5) By use of 20 μL of the HISCL washing liquid, washing by pipettingwas performed three times in total. (6) 20 μL of a 10 μg/mLstreptavidin/1%BSA/0.05%PBST solution was dropped and the resultantobject was left to stand for one hour at room temperature. (7) By use of20 μL of the HISCL washing liquid, washing by pipetting was performedfour times in total.

[Preparation of Other Substances]

Anti-human Amyloid β Mouse IgG (82E1) modified with biotin was preparedand was used as a first capture substance capable of binding to the baseplate. The prepared first capture substance corresponds to the firstcapture substance 50 of Embodiment 1. Magnetic beads having an anti-DNPantibody bound thereto (Anti-DNP labeled antibody labeled beads(manufactured by Sysmex Corporation)) were used as a solid phase. Theprepared solid phase corresponds to the solid phase 20 of Embodiment 1.Anti-human Amyloid β Mouse IgG (82E1) modified with DNP was prepared andwas used as a second capture substance capable of binding to the solidphase. The prepared second capture substance corresponds to the secondcapture substance 30 of Embodiment 1. Anti-human Amyloid β Mouse IgG(82E1) modified with a silyl rhodamine-based fluorescent dye wasprepared and was used as a fluorescence-labeled antibody. The preparedfluorescence-labeled antibody corresponds to the third capture substance40 of Embodiment 1. A 5 mM DNP-Lys. solution was used as a releaser. Theprepared releaser corresponds to the releaser used in Embodiment 1.

As the silyl rhodamine-based fluorescent dye, the one obtained throughsynthesis according to the description in the following document wasused. The document referenced in synthesizing the fluorescent dye wasJonathan B Grimm et al., “A general method to improve fluorophores forlive-cell and single-molecule microscopy”, nature methods, VOL.12 NO.3(2015) pp.244-250.

Procedure of Verification of Embodiment 1

(1) The first capture substance (Biotin-IgG), the second capturesubstance (DNP-IgG), and the fluorescence-labeled antibody (fluorescentdye-IgG) prepared as described above were mixed together, and themixture was adjusted with an HISCL R3 diluent (manufactured by SysmexCorporation) so as to have the composition as shown in the table below,whereby an antibody solution was prepared.

TABLE 1 Composition of antibody solution Antibody concentration Antibody(fmol/assay) Biotin-IgG 200 DNP-IgG 200 Fluorescent dye-IgG 200

(2) 80 μL of the antibody solution and 500 μL of the specimen were mixedtogether, and the mixture was allowed to react for 30 minutes at 37° C.(3) 20 μL of the solid phase was mixed thereto, and the resultantmixture was allowed to react for 15 minutes at 37° C. (4) The solidphase was collected by magnetic force, then the supernatant was removed(BF separation), and then, 20 μL of the HISCL washing liquid was added,and the resultant mixture was agitated. This step was performed threetimes in total. (5) After the BF separation, 10 μL of the 5 mM DNP-Lys.solution serving as the releaser was added, and the resultant mixturewas agitated. This mixture was allowed to react for 10 minutes at 37° C.(6) After the BF separation, the supernatant was collected, and wasdropped on the base plate. The resultant object was left to stand fortwo hours at room temperature. (7) By use of 20 μL of the HISCL washingliquid, washing by pipetting was performed three times in total. (8) Theinformation obtaining step similar to that shown in FIG. 12 wasperformed by use of a super-resolution fluorescence microscope, and thetest substance on the base plate was observed. Then, a super-resolutionimage was obtained.

PROCEDURE OF VERIFICATION OF COMPARATIVE EXAMPLE

(1) The first capture substance (Biotin-IgG) prepared as described abovewas adjusted with the HISCL R3 diluent so as to attain 200 fmol/assay,and 80 μL of the resultant mixture was added to a base plate and wasallowed to react for 30 minutes at 37° C. (2) After the supernatant wasremoved, washing was performed by use of 20 μL of the HISCL washingliquid. This step was performed three times in total. (3) 500 μL of thespecimen was added, and the resultant mixture was allowed to react for60 minutes at 37° C. (4) After the supernatant was removed, washing wasperformed by use of 20 μL of the HISCL washing liquid. This step wasperformed three times in total. (5) The fluorescence-labeled antibody(fluorescent dye-IgG) was adjusted with the HISCL R3 diluent so as toattain 200 fmol/assay, 80 μL of the resultant mixture was added to thebase plate, and the resultant mixture was allowed to react for 60minutes at 37° C. (6) After the supernatant was removed, washing wasperformed by use of 20 μL of the HISCL washing liquid. This step wasperformed three times in total. (7) The information obtaining stepsimilar to that shown in FIG. 12 was performed by use of asuper-resolution fluorescence microscope, and the test substance on thebase plate was observed. Then, a super-resolution image was obtained.

With reference to FIGS. 16A to 16C, the verification result ofEmbodiment 1 is described.

FIG. 16A shows super-resolution images obtained through the procedure ofverification of Embodiment 1. FIG. 16A shows super-resolution imagesobtained when arbitrary concentrations of the test substance in thespecimen were employed. As shown in FIG. 16A, due to the resolutionexceeding the diffraction limit, in each of the four super-resolutionimages, the shape of an aggregate of the test substance about 100 nm isdisplayed in a recognizable manner. Thus, according to Embodiment 1, avery small shape of the test substance exceeding the diffraction limitcan be observed, and thus, it is considered that information regardingthe structure of the test substance can be accurately obtained.

FIG. 16B shows fluorescence images each obtained by performing imagecapturing of fluorescence excited from all of the fluorescent dyes 41immediately after the start of step S101 in the information obtainingstep, in the procedure (8) of verification of Embodiment 1. The threefluorescence images shown in FIG. 16B are, from the left in order, thefluorescence images obtained when the concentrations of test substancein the specimen were 0 pM, 0.3 pM, and 1 pM, respectively. FIG. 16C is agraph showing the relationship between the number of bright spots on thethree fluorescence images shown in FIG. 16B and the concentration of thetest substance. As to the counting of the bright spots, each regionhaving a brightness exceeding a predetermined threshold in afluorescence image was defined as a bright spot region, and the numberof specified bright spot regions was counted.

As shown in FIG. 16C, in Embodiment 1, when the concentration of thetest substance is 0 pM, the number of bright spots is close to 0, and inaccordance with increase in concentration, the number of bright spotsincreased. Thus, according to Embodiment 1, even when the concentrationof the test substance is low, specific fluorescence detection regardingthe test substance can be realized, while influence of impurities issuppressed.

With reference to FIGS. 17A to 17C, the verification result ofComparative Example is described.

FIG. 17A shows a super-resolution image obtained through the procedureof verification of Comparative Example. FIG. 17A is a super-resolutionimage obtained when the concentration of the test substance in thespecimen was 0 pM. As shown in FIG. 17A, in Comparative Example,although the concentration of the test substance was 0 pM, a pluralityof bright spots exist, and thus, it is seen that there is much noiselight based on impurities. Therefore, in Comparative Example, sinceimpurities are mixed with the test substance on the base plate, cleardistinction between the test substance and the impurities are notrealized, and thus, information regarding the structure of the testsubstance becomes difficult to be accurately obtained.

FIG. 17B shows fluorescence images each obtained by performing imagecapturing of fluorescence excited from all of the fluorescent dyes 41immediately after the start of step S101 in the information obtainingstep, in the procedure (7) of verification of Comparative Example. Thethree fluorescence images shown in FIG. 17B are, from the left in order,the fluorescence images obtained when the concentrations of the testsubstance in the prepared specimen were 0 pM, 0.3 pM, and 1 pM,respectively. FIG. 17C is a graph showing the relationship between thenumber of bright spots on the three fluorescence images shown in FIG.17B and the concentration of the test substance. The counting of thebright spots was performed in a similar manner to that in theverification of Embodiment 1.

As shown in FIG. 17C, in Comparative Example, although the concentrationof the test substance is 0 pM, several thousands of bright spots exist,and the number of bright spots has not increased in accordance withincrease in concentration. Therefore, according to Comparative Example,due to influence of noise light based on impurities, specificfluorescence detection regarding the test substance is difficult.

Through the verifications above, it was found that, according toEmbodiment 1, information regarding the structure of the test substancecould be accurately obtained while influence of the impurities wassuppressed by removing the impurities.

<Detection Apparatus>

As shown in FIG. 18, the detection apparatus 100 includes an informationobtaining unit 101 and an information processing unit 102. The detectionapparatus 100 is an apparatus for automatically performing each step inthe information obtaining step shown in FIG. 12.

The information obtaining unit 101 includes a light source unit 110, ashutter 121, a ¼ wave plate 122, a beam expander 123, a condenser lens124, a dichroic mirror 125, an objective lens 126, a condenser lens 127,a stage 130, an image capturing unit 140, and controllers 151, 152. Onthe stage 130, the base plate 80 having the test substance 11immobilized thereon is set.

The light source unit 110 includes a light source 111 and a mirror 112.The light source 111 emits excitation light. As the light source 111, alaser light source is preferably used, but a mercury lamp, a xenon lamp,an LED, or the like may be used. The excitation light emitted from thelight source 111 changes the state of the fluorescent dye 41 bound tothe test substance 11 between the light emitting state and the quenchedstate, and causes the fluorescent dye 41 in the light emitting state tobe excited to generate fluorescence. The mirror 112 reflects theexcitation light from the light source 111 to guide the excitation lightto the shutter 121.

In a case where the fluorescent dye 41 is configured to be switchedbetween an active state in which the fluorescent dye 41 generatesfluorescence and an inactive state in which the fluorescent dye 41 doesnot generate fluorescence, the light source unit 110 is configured toinclude two light sources, a mirror, and a dichroic mirror. In thiscase, one of the light sources emits light that causes the fluorescentdye 41 to enter the active state, and the other of the light sourceemits light that causes the fluorescent dye 41 to enter the inactivestate. The optical axes of lights from the two light sources are causedto be aligned with each other by the mirror and the dichroic mirror.

The shutter 121 is driven by the controller 151, and performs switchingbetween a state in which the excitation light emitted from the lightsource unit 110 is allowed to pass therethrough, and a state in whichthe excitation light emitted from the light source unit 110 is blocked.Accordingly, the time period of the application of the excitation lightonto the test substance 11 is adjusted. The ¼ wave plate 122 convertsthe excitation light, which is linearly polarized light, emitted fromthe light source unit 110 into circularly polarized light. Thefluorescent dye 41 reacts with excitation light in a predeterminedpolarization direction. Thus, by converting the excitation light emittedfrom the light source unit 110 into circularly polarized light, thepolarization direction of the excitation light can be easily alignedwith the polarization direction in which the fluorescent dye 41 reacts.Accordingly, the fluorescent dye 41 can be efficiently excited togenerate fluorescence. The beam expander 123 widens the applicationregion of the excitation light on the base plate 80. The condenser lens124 collects the excitation light such that collimated light is appliedfrom the objective lens 126 to the base plate 80.

The dichroic mirror 125 reflects the excitation light emitted from thelight source unit 110, and allows fluorescence generated from thefluorescent dye 41 to pass therethrough. The objective lens 126 guidesto the base plate 80 the excitation light reflected by the dichroicmirror 125. The stage 130 is driven by the controller 152 so as to bemoved in the planar direction. Fluorescence generated from thefluorescent dye 41 on the base plate 80 passes through the objectivelens 126 and the dichroic mirror 125. The condenser lens 127 collectsfluorescence that has passed through the dichroic mirror 125 and guidesthe fluorescence to a light receiving surface 141 of the image capturingunit 140. The image capturing unit 140 captures an image of fluorescenceapplied on the light receiving surface 141, and generates a fluorescenceimage. The image capturing unit 140 is implemented by a CCD, forexample.

The information processing unit 102 includes a processing unit 161, astorage unit 162, a display unit 163, an input unit 164, and aninterface 165.

The processing unit 161 is a CPU, for example. The storage unit 162 is aROM, a RAM, a hard disk, or the like. On the basis of programs stored inthe storage unit 162 and through the interface 165, the processing unit161 controls each unit in the information processing unit 102, the lightsource 111 of the light source unit 110, the image capturing unit 140,and the controllers 151, 152.

On the basis of programs stored in the storage unit 162, the processingunit 161 performs the information obtaining step shown in FIG. 12. Thatis, in the information obtaining step, the processing unit 161 drivesthe light source 111 and causes the image capturing unit 140 to receivefluorescence generated from the fluorescent dye 41, and drives the imagecapturing unit 140 to obtain fluorescence images. On the basis of thefluorescence images obtained by the image capturing unit 140, theprocessing unit 161 generates a super-resolution image. On the basis ofthe generated super-resolution image, the processing unit 161 obtainsinformation regarding the structure of the test substance 11, and causesthe display unit 163 to display a screen including the obtainedinformation.

The display unit 163 is a display for displaying a process result andthe like obtained by the processing unit 161. The display unit 163displays the screen 90 shown in FIG. 15. The input unit 164 is akeyboard and a mouse for receiving inputs of instructions from anoperator.

<Modification>

In the information obtaining step shown in FIG. 12, the test substance11 on the base plate 80 is measured by means of a super-resolutionfluorescence microscope having a resolution exceeding the diffractionlimit of light. However, not limited thereto, the test substance 11 onthe base plate 80 may be measured by means of a Raman microscope, aprobe microscope, or an electron microscope. With a probe microscope andan electron microscope, the test substance 11 can be measured at aresolution exceeding the diffraction limit of light. In a case wheremeasurement by use of fluorescence is not performed in the informationobtaining step, for example, in a case where a Raman microscope or aprobe microscope is used, the labeling step described above is omitted.When the labeling step is omitted, it becomes easy to cause the firstcapture substance 50 to bind to the binding site of the test substance11, and thus, the test substance 11 can be more smoothly immobilized onthe base plate 80.

The size, the morphology, and the aggregation degree regarding the testsubstance 11 in the information regarding the structure of the testsubstance 11 can be obtained if the test substance 11 is measured by useof a super-resolution fluorescence microscope, a Raman microscope, aprobe microscope, or an electron microscope. The structure of the testsubstance 11 in the information regarding the structure of the testsubstance 11 can be obtained if the test substance 11 is measured by useof a super-resolution fluorescence microscope, a Raman microscope, or aprobe microscope.

When the test substance 11 is measured by use of a Raman microscope,Raman spectra reflecting the molecules or the atoms which form the testsubstance 11, and an image reflecting the shape of the test substance 11are obtained. Thus, according to a Raman microscope, as informationregarding the structure of the test substance 11, the chemical bond canalso be obtained in addition to the size, the morphology, the structure,and the aggregation degree. Specifically, as the chemical bond of thetest substance 11, the kind, number, concentration, proportion, and thelike regarding the molecules or the atoms which form the test substance11 are obtained. In this case, in the region 93 of the screen 90 shownin FIG. 15, the obtained chemical bond of the test substance 11 isdisplayed, in addition to the size, the morphology, the structure, andthe aggregation degree. For example, in the region 93, as the obtainedchemical bond of the test substance 11, “C═0 is at a concentration of .. . , C—H is at a concentration of . . . ”, etc. is displayed.

What is claimed is:
 1. A method for obtaining information of a testsubstance, the method comprising: forming a complex by causing a capturesubstance to bind to a test substance in a specimen; selectivelycollecting at least the complex from the specimen; immobilizing thecomplex collected from the specimen, onto a base plate; and obtaininginformation regarding a structure of the test substance from the compleximmobilized on the base plate.
 2. The method for obtaining theinformation of the test substance of claim 1, wherein in the obtainingof the information regarding the structure of the test substance, atleast one of size, morphology, structure, and aggregation degree of thetest substance is obtained.
 3. The method for obtaining the informationof the test substance of claim 1, wherein the capture substance includesa capture substance for labeling the test substance with fluorescence.4. The method for obtaining the information of the test substance ofclaim 1, wherein the capture substance includes a capture substancecapable of binding to a solid phase.
 5. The method for obtaining theinformation of the test substance of claim 1, wherein the capturesubstance includes a capture substance capable of binding to the baseplate.
 6. The method for obtaining the information of the test substanceof claim 3, wherein the capture substance for labeling the testsubstance with fluorescence includes an antibody labeled with afluorescent dye; and in the forming of the complex, the antibody labeledwith the fluorescent dye is caused to bind to the test substance.
 7. Themethod for obtaining the information of the test substance of claim 4,wherein after the solid phase is caused to bind to the complex throughthe capture substance capable of binding to the solid phase, the solidphase is selectively separated in the collecting of the complex from thespecimen.
 8. The method for obtaining the information of the testsubstance of claim 7, wherein in the collecting of the complex from thespecimen, the complex is caused to be detached from the solid phaseafter the solid phase is selectively separated.
 9. The method forobtaining the information of the test substance of claim 7, wherein thesolid phase includes a magnetic particle, and the solid phase isselectively separated by attracting the magnetic particle by magneticforce.
 10. The method for obtaining the information of the testsubstance of claim 7, wherein the capture substance capable of bindingto the solid phase includes an antibody which binds to the testsubstance and a second binding substance which binds to the solid phase,the solid phase includes a second binding partner which specificallybinds to the second binding substance, and binding between the complexand the solid phase is realized by binding between the test substanceand the antibody of the capture substance capable of binding to thesolid phase and by specific binding between the second binding substanceand the second binding partner.
 11. The method for obtaining theinformation of the test substance of claim 1, wherein the capturesubstance includes a capture substance capable of binding to the baseplate and a capture substance capable of binding to a solid phase, andthe capture substance capable of binding to the base plate and thecapture substance capable of binding to the solid phase are differentfrom each other.
 12. The method for obtaining the information of thetest substance of claim 1, wherein in the collecting of the complex fromthe specimen, the complex is separated from an impurity on the basis ofat least one of a difference in specific gravity, a difference in size,a difference in electrical property, and a difference in immunoreactionbetween the complex and the impurity.
 13. The method for obtaining theinformation of the test substance of claim 5, wherein the capturesubstance capable of binding to the base plate includes an antibodywhich binds to the test substance and a binding substance which binds tothe base plate, the base plate includes a binding partner whichspecifically binds to the binding substance, and after the antibodyincluded in the capture substance capable of binding to the base plateis caused to bind to the test substance, the complex is immobilized onthe base plate through specific binding between the binding partner andthe binding substance, in the immobilizing of the complex onto the baseplate.
 14. The method for obtaining the information of the testsubstance of claim 1, wherein after the collecting of the complex fromthe specimen, a capture substance capable of binding to the base plateis caused to bind to the test substance.
 15. The method for obtainingthe information of the test substance of claim 1, wherein in theobtaining of the information regarding the structure of the testsubstance, an image of the test substance is obtained by performingimage capturing of the test substance on the base plate.
 16. The methodfor obtaining the information of the test substance of claim 1, whereinin the collecting of the complex from the specimen, the test substanceand an impurity are separated from each other, and the test substance isa protein as a test target contained in the specimen and the impurityincludes a protein other than the protein as the test target.
 17. Themethod for obtaining the information of the test substance of claim 1,wherein the test substance is amyloid β.
 18. The method for obtainingthe information of the test substance of claim 1, wherein the obtainingof the information regarding the structure of the test substance isperformed by means of a microscope having a resolution exceeding adiffraction limit of light.
 19. A method for obtaining information of atest substance, the method comprising: causing a magnetic particle tobind to a test substance in a specimen; selectively collecting at leastthe test substance from the specimen by use of the magnetic particlebound to the test substance; immobilizing the test substance collectedfrom the specimen, onto a base plate; and obtaining informationregarding a structure of the test substance from the test substanceimmobilized on the base plate.
 20. A method for obtaining information ofa test substance, the method comprising: forming a complex by causing acapture substance including a fluorescent dye to bind to a testsubstance in a specimen; selectively collecting at least the complexfrom the specimen; immobilizing the complex collected from the specimen,onto a base plate; and obtaining information regarding a structure ofthe test substance on the basis of bright spots corresponding tofluorescence emitted from a plurality of fluorescent dyes each bound tothe complex immobilized on the base plate.